African Journal of Biotechnology Vol. 7 (13), pp. 2232-2238, 4 July, 2008 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2008 Academic Journals Full Length Research Paper Bacterial removal of toxic phenols from an industrial effluent J. Lin*, M. Reddy, V. Moorthi and B. E. Qoma School of Biochemistry, Genetics, Microbiology and Plant Pathology, University of KwaZulu-Natal (Westville), Private Bag X 54001, Durban, Republic of South Africa. Accepted 28 April, 2008 Chlorinated phenols, widely used in industries, are of growing concern owing to their high toxicity, carcinogenicity and wide distribution in industrial wastes. In the present study, one Pseudomonas isolate, identified as Pseudomonas fluorescens, was obtained using the enrichment process with 2,4,6- trichlorophenol (2,4,6-TCP) as a sole carbon source. This isolate was found to be able to degrade various highly chlorinated phenolic compounds such as pentachlorophenol, 2,4,5-TCP, 2,4,6-TCP as well as phenol, 2,4-dibromophenol and 2,4-dichlorophenol (2,4-DCP). The ability of P. fluorescens isolate to remove phenol from a resin producing industrial effluent was tested by scanning the spectrum with a UV-VIS spectrophotometer. The results indicated that this isolate metabolized phenol in the meta-pathway. The optimal phenol degradation conditions of P. fluorescens isolate were at pH 7 and 30oC. At the 480 mg/l of phenol concentration, the highest specific degradation rate of was observed. Further increases in phenol concentration slowed down the degradation ability of the isolate. However, P. fluorescens isolate still has the ability of degrading phenol at the concentration of 3.2 g/L. The supplementation of 1% glucose stimulated the growth of P. fluorescens isolate and enhanced the ability to utilize phenol from the effluent sample. GC-MS results show that 85.4% of phenol in the effluent sample was metabolized after 40 days. In conclusion, P. fluorescens isolated in this study has the ability of utilizing various chlorophenolic compounds and demonstrates its potentials of degrading high concentration of phenol in industrial effluents. Key words: Bioremediation, Pseudomonas fluorescens, industrial effluent, chlorophenols. INTRODUCTION Chlorinated phenols are important chemicals widely used (Exon, 1984). Owing to their high toxicity, carcinogenicity in industries for the manufacture of products such as and wide distribution in industrial wastes leading to great dyes, insecticides, disinfectants, wood preservatives, as harm to human being and marine organisms (Exon, chemical products in building, agriculture and hospital 1984; Ping et al., 2003), they are of growing concern (Hoos, 1978; Haggblom, 1992; Agostini et al., 2003) and (Alexander, 1981; Sittig, 1981). in effluents from pulp and paper mills (Watanabe et al., Although there have been many studies regarding the 1996). Because of its broad application in industrial and environment fate of chlorinated phenols, including medical settings, it has become one of environmental photochemical degradation and sequential aerobic and contaminants especially in the underground water anaerobic degradation. The bioremediation means have (Radchaus and Schmidt, 1992; Breining et al., 2000). been generally accepted by the public because they lead Chlorophenols stick to soil and to sediments at the innocuous tailings, are non-invasive to environments and bottom of lakes, rivers, or streams and rapidly enter the are cost-efficient compared physiochemical approaches body through the skin and the gastrointestinal tract (Gallizia et al., 2003). Biodegradation is defined as the biologically catalyzed reduction in complexity of chemical compounds. The organic pollutants are used as sole source of carbon and energy. Growth process results in a *Corresponding author. E-mail: [email protected]. Tel: +27-31- complete degradation (mineralization) of organic 2607401. Fax: +27-31-2607809. pollutants. Generally chlorinated phenols are transformed Lin et al. 2233 via oxidative dechlorination (aerobic biodegradation) Table 1. The substrate preference of P. fluorescens isolate (Steinle et al., 1998), while in anaerobic conditions via using various halo-phenolic compounds as the sole carbon reductive dechlorination (Annachhatre and Gheewala, source in BH media at pH7.2, 30ºC and 160 rpm for 4 days. 1996). In the present study, we report the characteristics of Substrate P. fluorescens Pseudomonas fluorescens isolate capable of degrading Phenol (200 M) + + + various chlorophenolic compounds and the potentials to 2,4-dibromophenol (200 M) + + + + remove the phenolic compounds from a local resin 2,4-dichlorophenol (200 M) + + + + producing industrial effluent. 4-chloro-3-nitrophenol (200 M) + + + + 2,6-dichlorophenol (200 M) + + + + + + + + + + MATERIALS AND METHODS 2,4,6-trichlorophenol (200 M) 2,4,5-trichlorophenol (200 M) + + Isolation and identification Pentachlorophenol (200 M) + + Soil samples were collected from Botany nursery (garden) at the + + + + +: OD 600 > 0.700. Westville campus of the University of KwaZulu Natal. 10g of garden + + +: 0.700> OD 600 >0.400. soil was added into 100 ml of Bushnell Haas (BH) medium, pH7.2, + +: 0.400> OD 600 > 0.200. containing NH NO (1 g/L), K HPO (1 g/L), KH PO (1 g/L), +: OD 600 <0.200. 4 3 2 4 2 4 CaCl .2H O (0.02 g/L), MgSO .7H O (0.2 g/L), FeCl (0.0 5g/L) 2 2 4 2 3 (Atlas, 1994). 200 M of 2,4,6-trichlorophenol (2,4,6-TCP) was o used as sole carbon source. These flasks were shaken at 30 C and using TLC and the colorimetric methods, while the phenol concen- 160 rpm for four weeks with 1 ml of enriched media transferred into tration was high, as described below. The cell growth was also freshly prepared enrichment media each week. Serial dilutions monitored by optical density at 600 nm (OD600). (1/10) of final enriched media were spread-plated on BH agar o supplemented with 2,4,6-TCP incubated at 30 C. The single colo- nies were streaked onto nutrient agar plates, incubated at 30oC o Determination of phenol concentration overnight and the pure isolates were stored at 4 C until further use. The isolate that was capable of degrading chlorophenolic com- 1 ml of supernatant after centrifugation at given time point was pounds were identified by Gram stain, biochemical tests (Garrity et assayed by scanning its spectrum at 200-400 nm with a Cary 50 UV al., 2005) and confirmed by 16S rDNA sequencing (Marchesl et al., spectrophotometer (Varian Inc.). The concentration of phenol was 1998). For long–term preservation, the bacterial isolate was stored determined by OD268 with the absorbance coefficient of 2169 (El- o in 40% glycerol at -70 C. Sayed et al., 2003). Substrate preferences for P. fluorescens isolate Thin Layer chromatography (TLC) The substrate preference of the isolate was studied by using The aliquots of treated effluents were spotted on a TLC plate various chlorophenolic compounds as carbon sources (Table 1) at (Merck Silica gel 60 F254). The solvent system was toluene, acetyl 30oC and 160 rpm. The cell growth was measured by the optical acetate and acetic acid in 7:2:1 ratio (Harborne, 1986). The pheno- density at 600 nm. lic compound was visualized using Syngene Genius gel documen- tation system. The concentrations of phenolic compound was measured based on the total integrated area of the designated spot Degradation of phenolic compounds in an industrial effluent compared with the control (Rf value of 0.57). One industrial effluent containing phenolic compounds from a local resin producing plant was used as the sole carbon source to test Colorimetric assay of phenolic compounds the degradation potentials of the bacterial isolate. During the course The residual amounts of phenolic compounds present in the of the study, phenol (32 g/l) was identified as the main component in the effluent using GC-MS and colorimetric analyses. effluents at different incubation period were also measured by colorimetric assay (Klibanov et al., 1980). In brief, aliquots (5 ml) of The bacterial isolate was grown overnight in nutrient broth and the grown bacteria were centrifuged for 15 min at 3000 rpm. Cells sample were reacted with 0.025 ml of 4-aminoantipyrine (2% aqueous solution), 0.025 ml of 6.0 M NH4OH and 0.05 ml potas- were re-suspended on BH broth. Cell number of each isolate was sium ferricyanide (8% w/v). After 5 min, the dye formed was normalized to optical density (OD ) of 1. 600 extracted with 2.5 ml of chloroform. The absorbance of the extract Preliminary degradation studies were carried out with the addition was determined at 510 nm. of 1 ml normalized inocula into 100 ml of BH broth containing 160 mg/l of phenol from the industrial effluent. Flasks were shaken at 30oC and 160 rpm. The reactions containing all components but devoid of bacterial inoculums were used as the controls. Optimal Gas chromatography- mass spectrometry (GC-MS) conditions for the substrate degradation by bacterial isolate were The samples during bacterial treatment were analyzed using GC- determined using different pHs (pH 6.0, 7.2 and 9.0), temperatures MS. Agilent 6890 GC/5973 MS with J and W HP5-MS column (300 and different concentrations of substrate. In order to enhance the x 2.5 mm) was used. phenol-degradation ability of bacterial isolate at the high phenol concentration, additional 1% glucose was supplemented in the growth media. Calculation of specific degradation rate The phenol concentration was determined using a UV-VIS spec- trophotometer (Varian Cary 50) at low phenol concentration and The specific phenol degradation rate of P. fluorescens isolate under 2234 Afr. J. Biotechnol. A B Day 0 Day 0 Day 2 Day 3 Day 2 Day 3 2-HMS Figure 1. Phenol biodegradation measured by scanning the spectrum with a UV-VIS spectrophotometer at different time intervals in the absence (A) and the presence (B) of P. fluorescens isolate.